TVScript: A Tool for Exploring the Effects of Intra-Condition Variation on the Detection of Differentially Expressed Genes

The evolution of RNA-Seq technologies yielded datasets that are of immense scientific value. Commonly, such data is generated within differential expression studies, where datasets derived from individual samples are grouped into conditions, and gene expression patterns quantified. The number of archived datasets is increasing and revisiting many at an inter-study level provides an in-depth view into transcriptome evolution. The biggest hurdle is in dealing with variation of read counts at an individual transcript level between common conditions. We present a tool, TVScript, that quantifies intra-condition variation, and subsequently, removes reference-based transcripts that are associated with high levels of this. TVScript is demonstrated at inter and intra-study levels, using data from brain samples of dogs, wolves and foxes (aggressive and tame), where a marked improvement in the distribution of the gene-wise dispersion estimates, the metric utilized by the majority of differential expression tools, lowered the number of outliers detected. We provide support for seven candidate genes with potential for being involved with selection for tameness, and that appear to play a crucial role in canine domestication. We also identify several genes previously identified as being differentially expressed, but that possessed high intra-condition variation, weakening their relevance. TVScript is available at: https://sourceforge.net/projects/tvscript/.

HIV-1: To Splice or Not to Splice, That Is the Question

The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

TREND-DB—a transcriptome-wide atlas of the dynamic landscape of alternative polyadenylation

Alternative polyadenylation (APA) profoundly expands the transcriptome complexity. Perturbations of APA can disrupt biological processes, ultimately resulting in devastating disorders. A major challenge in identifying mechanisms and consequences of APA (and its perturbations) lies in the complexity of RNA 3′ end processing, involving poorly conserved RNA motifs and multi-component complexes consisting of far more than 50 proteins. This is further complicated in that RNA 3′ end maturation is closely linked to transcription, RNA processing and even epigenetic (histone/DNA/RNA) modifications. Here, we present TREND-DB (http://shiny.imbei.uni-mainz.de:3838/trend-db), a resource cataloging the dynamic landscape of APA after depletion of >170 proteins involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications and further processes. TREND-DB visualizes the dynamics of transcriptome 3′ end diversification (TREND) in a highly interactive manner; it provides a global APA network map and allows interrogating genes affected by specific APA-regulators and vice versa. It also permits condition-specific functional enrichment analyses of APA-affected genes, which suggest wide biological and clinical relevance across all RNAi conditions. The implementation of the UCSC Genome Browser provides additional customizable layers of gene regulation accounting for individual transcript isoforms (e.g. epigenetics, miRNA-binding sites and RNA-binding proteins). TREND-DB thereby fosters disentangling the role of APA for various biological programs, including potential disease mechanisms, and helps identify their diagnostic and therapeutic potential.

TREND-DB – A Transcriptome-wide Atlas of the Dynamic Landscape of Alternative Polyadenylation

Alternative polyadenylation (APA) profoundly expands the transcriptome complexity. Perturbations of APA can disrupt biological processes, ultimately resulting in devastating disorders. A major challenge in identifying mechanisms and consequences of APA (and its perturbations) lies in the complexity of RNA 3’end processing, involving poorly conserved RNA motifs and multi-component complexes consisting of far more than 50 proteins. This is further complicated in that RNA 3’end maturation is closely linked to transcription, RNA processing, and even epigenetic (histone/DNA/RNA) modifications. Here we present TREND-DB (http://shiny.imbei.uni-mainz.de:3838/trend-db), a resource cataloging the dynamic landscape of APA after depletion of >170 proteins involved in various facets of transcriptional, co- and posttranscriptional gene regulation, epigenetic modifications, and further processes. TREND-DB visualizes the dynamics of transcriptome 3’end diversification (TREND) in a highly interactive manner; it provides a global APA network map and allows interrogating genes affected by specific APA-regulators, and vice versa. It also permits condition-specific functional enrichment analyses of APA-affected genes, which suggest wide biological and clinical relevance across all RNAi conditions. The implementation of the UCSC Genome Browser provides additional customizable layers of gene regulation accounting for individual transcript isoforms (e.g. epigenetics, miRNA binding sites, RNA-binding proteins). TREND-DB thereby fosters disentangling the role of APA for various biological programs, including potential disease mechanisms, and helps to identify their diagnostic and therapeutic potential.

Ribosome profiling at isoform level reveals an evolutionary conserved impact of differential splicing on the proteome

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Yet, its impact in protein production remains an open question. Here, we describe ORQAS (ORF quantification pipeline for alternative splicing), a new pipeline for the translation quantification of individual transcript isoforms using ribosome-protected mRNA fragments (Ribosome profiling). We found evidence of translation for 40-50% of the expressed transcript isoforms in human and mouse, with 53% of the expressed genes having more than one translated isoform in human, 33% in mouse. Differential analysis revealed that about 40% of the splicing changes at RNA level were concordant with changes in translation, with 21.7% of changes at RNA level and 17.8% at translation level conserved between human and mouse. Furthermore, orthologous cassette exons preserving the directionality of the change were conserved between human and mouse and enriched in microexons in a comparison between glia and glioma. ORQAS leverages ribosome profiling to uncover a widespread and evolutionary conserved impact of differential splicing on the translation of isoforms and, in particular, of microexon-containing isoforms. ORQAS is available at https://github.com/comprna/orqas

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs

SummaryHuman induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across nine time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells that were validated using electrophysiology. We identified widespread changes in the expression of individual transcript features and their splice variants, gene networks, and global patterns of transcription. We next demonstrated that co-culturing human NPCs with rodent astrocytes resulted in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without potentially disruptive cell sorting. We lastly developed and validated a computational tool to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue, which were maturationally heterogeneous but contained subsets of cells most akin to adult human neurons.

Transcriptional profiling of macrophages derived from monocytes and iPS cells identifies a conserved response to LPS and novel alternative transcription

Macrophages differentiated from human induced pluripotent stem cells (IPSDMs) are a potentially valuable new tool for linking genotype to phenotype in functional studies. However, at a genome-wide level these cells have remained largely uncharacterised. Here, we compared the transcriptomes of naïve and lipopolysaccharide (LPS) stimulated monocyte-derived macrophages (MDMs) and IPSDMs using RNA-Seq. The IPSDM and MDM transcriptomes were broadly similar and exhibited a highly conserved response to LPS. However, there were also significant differences in the expression of genes associated with antigen presentation and tissue remodelling. Furthermore, genes coding for multiple chemokine involved in neutrophil recruitment were more highly expressed in IPSDMs upon LPS stimulation. Additionally, analysing individual transcript expression identified hundreds of genes undergoing alternative promoter and 3′ untranslated region usage following LPS treatment representing a previously under-appreciated level of regulation in the LPS response.